Phylogenetic and expression analysis of the NPR1-like gene family from Persea americana (Mill.)

Abstract

The NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) forms an integral part of the salicylic acid (SA) pathway in plants and is involved in cross-talk between the SA and jasmonic acid/ethylene (JA/ET) pathways. Therefore, NPR1 is essential to the effective response of plants to pathogens. Avocado (Persea americana) is a commercially important crop worldwide. Significant losses in production result from Phytophthora root rot, caused by the hemibiotroph, Phytophthora cinnamomi. This oomycete infects the feeder roots of avocado trees leading to an overall decline in health and eventual death. The interaction between avocado and P. cinnamomi is poorly understood and as such limited control strategies exist. Thus uncovering the role of NPR1 in avocado could provide novel insights into the avocado - P. cinnamomi interaction. A total of five NPR1-like sequences were identified. These sequences were annotated using FGENESH and a maximum-likelihood tree was constructed using 34 NPR1-like protein sequences from other plant species. The conserved protein domains and functional motifs of these sequences were predicted. Reverse transcription quantitative PCR was used to analyze the expression of the five NPR1-like sequences in the roots of avocado after treatment with salicylic and jasmonic acid, P. cinnamomi infection, across different tissues and in P. cinnamomi infected tolerant and susceptible rootstocks. Of the five NPR1-like sequences three have strong support for a defensive role while two are most likely involved in development. Significant differences in the expression profiles of these five NPR1-like genes were observed, assisting in functional classification. Understanding the interaction of avocado and P. cinnamomi is essential to developing new control strategies. This work enables further classification of these genes by means of functional annotation and is a crucial step in understanding the role of NPR1 during P. cinnamomi infection.

Keywords: NPR1; Phytophthora cinnamomi; avocado; expression analysis; jasmonic acid; pathogenesis-related; salicylic acid.

FIGURE 1

Exon/intron boundary and predicted coding sequence comparison of PaNPR1-like genes with other known NPR1-like sequences. (A) The predicted exon/intron structure of the NPR1-like family from Arabidopsis thaliana and Persea americana. Exons are denoted by yellow boxes while introns are represented by thin black lines. (B) A comparison of the positions of the BTB/POZ and ankyrin repeat domains between the PaNPR1-like and AtNPR1-like family of proteins. (C) A multiple alignment of PaNPR1-like proteins and several other known NPR1-like proteins from woody plants and Arabidopsis. The positions of amino acid changes causing the npr1-1(H), npr1-2 (C), npr1-3 (^∗), and nim1-4 (R) mutants as well as the positions of the highly conserved cysteine residues at position 82 and 216 in Arabidopsis are indicated by black triangles above the alignment. The BTB/POZ and ankyrin repeat domains are indicated by black bars below the alignment. Several important motifs such as the IκB phosphodegron, LENRV hinge region, NIMIN1/2 binding site, and NLS1, are also indicated by black bars. The positions of important amino acids in the NLS1 of AtNPR1 are indicated by black dots above the alignment.

FIGURE 2

Phylogenetic analysis of the NPR1-like family of proteins from P. americana and several other species. A phylogenetic tree of five NPR1-like proteins from P. americana as well as NPR1-like proteins from other vascular and non-vascular plant species, including the NPR1-like family from Arabidopsis. The tree was generated in MEGA software v5.2 using the maximum likelihood (ML) method. A total of 1000 bootstrap replicates were performed and values are indicated above the branch points. The species of origin, identifiers and accession numbers are summarized in Table 1.

FIGURE 3

Expression of PaNPR1-like genes as well as PaPR1 in response to SA, JA, and Phytophthora cinnamomi treatment. Normalized relative quantities (fold change) for (A) PaNPR1, (B) PaNPR2, (C) PaNPR4, (D) PaNPR5, and (E) PaPR1 were calculated using the method described by Pfaffl (2001). The response is indicated by vertical bars across five time points for SA (green), MeJA (blue), and P. cinnamomi (red) treated samples, and is labeled on the horizontal axis. The samples were compared to untreated samples harvested at each time point, a representative bar for the controls (yellow) is also indicated. SE for each bar is shown. Significant differences (P < 0.05) between control and treated samples is denoted by an asterisk (^∗) above the bar.

FIGURE 4

Expression of PaNPR1-like genes in various tissues. Normalized relative quantities (fold change) for (A) PaNPR1, (B) PaNPR2, (C) PaNPR3, (D) PaNPR4, and (E) PaNPR5 were calculated using the method described by Pfaffl (2001). The response is indicated by vertical bars across six tissues; feeder roots, mature green stems, mature green leaves, unripe fruit as well as stems and leaves from flush growth (young material), and is labeled on the horizontal axis. The expression in all tissues was calibrated using expression in the roots. SE for each bar is shown. Bars represented with the same letter are not significantly different at P < 0.05.

FIGURE 5

Expression of PaNPR1-like genes in tolerant (Dusa^®) and susceptible (R0.12) avocado rootstocks infected with P. cinnamomi. Normalized relative quantities (fold change) for (A) PaNPR1, (B) PaNPR2, (C) PaNPR4, and (D) PaNPR5 were calculated using the method described by Pfaffl (2001). The response is indicated by horizontal lines (red – tolerant, green – susceptible) across three time points; 3, 6, and 24 h, labeled on the horizontal axis. The expression was calibrated using expression at 0 h (uninfected control) which was set to a normalized relative expression of 1. SE for each bar is shown. Significant differences (P < 0.05) between control and treated samples is denoted by an asterisk (*) above the data point.